UV sunlight induces chemical reactions in DNA which, if allowed to accumulate, give rise to mutations in skin cells and eventually lead to the development of skin cancer. One of the chief biological defenses against the carcinogenic effects of UV light on skin is Nucleotide Excision Repair (NER), a biologic pathway evolved to remove damage to DNA, including UV light-induced lesions. The molecular underpinnings of NER are being elucidated, but many questions remain regarding the order of events, particularly, the mechanisms by which the steps of NER progress. The specific aims of this proposal are to determine in an evolutionarily conserved eukaryotic model system, the yeast S. cerevisiae: (1) whether NER occurs in spatially localized regions within the nucleus, and the timing and order of recruitment of the proteins Radio, Rad14 and Rad23;(2) that the DNA damage recognition protein factor Rad14 and perhaps the Rad4/Rad23 complex are required prior to participation of the downstream protein Radio;and (3) whether key amino acid residues of the Rad1 protein are required for recruitment of the Rad1/Radio complex to an NER site. These questions will be answered using a novel technique in which the proteins are labeled and tracked in live yeast cells with the aid of a fluorescence microscope. Cells will be exposed to UV light, and the recruitment of the proteins to DNA repair centers will be monitored as a function of time by comparing appropriate combinations of labeled proteins and mutant genes to answer the above questions. In preliminary experiments, one of the proteins, Radio, has already been fluorescently labeled and tested for its action in NER;results suggest that NER occurs in spatially localized regions within the nucleus. These experiments are being carried out in the S. cerevisiae model system rather than human cells for technical reasons. However, due to the evolutionary conservation of the NER pathway, the findings will be transferable to the human system. Skin cancer is a growing health problem in the U.S. society as a result of increased human exposure levels to damaging UV sunlight. New advances in clinical cancer prevention and treatment are a vital part of addressing this growing problem. A more detailed understanding of the biochemistry by which cells repair DNA will aid in finding new potential drug targets for pharmaceuticals that might minimize skin cancer risk.